Despite major medical advances, diarrheal diseases still cause ~2.6 million deaths per year worldwide. Therefore, a better understanding of the pathophysiology of diarrheal diseases is critical for designing novel and superior strategies for interventions. Enteropathogenic E. coli (EPEC), a food-borne pathogen, is a major cause of infantile diarrhea worldwide causing high rate of morbidity and mortality. To date, however, the mechanisms underlying EPEC-induced diarrhea are not well understood. Diarrhea occurs either via increased intestinal secretion or decreased absorption, or both. In this regard, electroneutral NaCl absorption is the predominant route for Na+ and Cl- absorption in the human ileum and colon. Intestinal luminal membrane NHE3 (sodium hydrogen exchanger 3) and DRA [Down Regulated in Adenoma, SLC26A3 Cl-/HCO3-(OH-) exchanger] proteins play critical roles in electroneutral NaCl absorption in the human intestine. Earlier published and preliminary studies from our group demonstrated that the early events following EPEC-induced diarrhea involved inhibition of NHE3 and DRA activity due to their internalization from surface of intestinal epithelial cells (IECs) via distinct mechanisms. NHE3 inhibition required E. coli secreted protein EspF and host IEC PDZ-protein NHERF2, whereas internalization of DRA required EPEC secreted EspGs (G1/G2) and disruption of IEC microtubular network. Based on these data, we hypothesize that acute EPEC infection inhibits intestinal NaCl absorption via cellular internalization of cell surface NHE3 and DRA. This internalization requires complex interactions between EPEC-secreted proteins and host IEC lipid-rafts, cytoskeletal (ezrin, actin) and PDZ domain proteins (NHERFs). Therefore, the objective of our current studies is to explore in detail the trafficking mechanisms underlying EPEC-induced internalization of NHE3 and DRA in intestinal epithelial cells utilizing state-of-the art in vitro and in vivo approaches. Specific Aim 1 has been designed to yield critical mechanistic information on contribution of lipid rafts, cytoskeletal and NHERF proteins in EPEC and EspF- induced inhibition of NHE3 activity. Specific Aim 2 will investigate mechanisms underlying DRA trafficking in vitro, including the role of lipid rafts, microtubules and NHERF proteins in response to EPEC or EspGs. Specific Aim 3 will utilize in vivo mouse models to validate in vitro data by examining the effects of EPEC infection on NHE3 and DRA and net impact on coupled NaCl and water absorption utilizing state-of-the-art techniques, NHERF knockout mice and mice administered colchicine. Our proposed studies should provide new insights into the pathophysiology of EPEC-induced diarrhea and mechanisms underlying regulation of NaCl absorption in the mammalian intestine and are, therefore, of great physiological and clinical significance. It is likely that these studies may identify novel molecular targets involved in EPEC-IEC interactions culminating in impaired NaCl absorption and may aid in the future development of better management strategies for the treatment of infectious diarrhea. PUBLIC HEALTH RELEVANCE: Despite major advances in medical advancements, diarrheal diseases cause ~2.6 million deaths per year worldwide, out of which about 1.5 million deaths are seen in children below the age of 5. Therefore, a better understanding of the basis of diarrheal diseases is critical for designing new and superior strategies for the treatment. Diarrhea occurs via increased intestinal secretion, decreased absorption, or both. In this regard, enteropathogenic E. coli (EPEC), an important food borne bacteria is known to cause widespread disease and death in children worldwide. In spite of knowing about this disease for many decades, the mechanisms involved in diarrhea in response to infection by these bacteria had not been well understood. Recent studies from our group suggested that one of the important mechanisms for early events of EPEC mediated diarrhea involved a decrease in salt (NaCl) and water absorption. The proposed studies in this application are designed to understand the detailed mechanisms of this reduction in salt and water by studying the molecular and cellular events underlying this reduction in salt and water absorption in response to bacterial infection. It is expected that these detailed molecular and cellular studies will identify new targets for the development of better treatment protocols for diarrheal diseases in future.